Project Summary Lyme disease is an endemic tick-borne disease associated with debilitating manifestations such as arthritis, muscle pain, carditis, meningitis, and encephalomyelitis. Despite extensive efforts in the field, there is still no vaccine for the prevention of this infection available for human use. To address this problem, our laboratory has developed novel methodologies to identify antigens relevant during bacterial infection in ticks and mammals and design peptide antigens based on extracellular and conserved regions of these proteins. Our hypothesis for this work is that peptides based on outer-membrane proteins expressed in various phases of the enzootic cycle of B. burgdorferi can be used as protective vaccines antigens against Lyme disease. To test this, we propose to perform RNA sequencing of B. burgdorferi during infection of ticks and mice to identify the genes most highly expressed in both organisms (Aim 1). Our objective is to synthesize peptides based on the extracellular regions of these proteins, and determine their antigenicity in a murine model. We will select antigens that are highly conserved, immunogenic, and do not display cross-reactivity with human antigens to avoid off- target reactivity (Aim 2). We will then test these antigens in a murine model of vaccination and challenge in combination with various adjuvants capable of eliciting specific humoral and cellular immune responses. To do this, we will use various murine reporter systems for IFN-?, IL-6 and IL-17 to measure bacterial clearance and T-cell response during immunization and challenge (Aim 3). To select the best adjuvant, we will also measure germinal center formation and memory B and T cell production. At the conclusion of these studies, we will have formulated a peptide-based vaccine containing antigens based on outer-membrane proteins with an adjuvant capable of conferring a strong cellular immune response. These studies will help with the identification of novel antigens and provide answers for the prevention of Lyme disease.